List of Titles

Identification of novel Arf1 GTPase inhibitors for cancer target validation

Authors: Mqwathi, Nomxolisi Vuyokasi
Date: 2023-10-13
Subjects: ARF1 , GTPase , Guanosine triphosphatase , Cancer Treatment
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/424666 , vital:72173
Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites

- Du Toit, Skye Carol

Authors: Du Toit, Skye Carol
Date: 2023-10-13
Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/424654 , vital:72172
Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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Investigating assay formats for screening malaria Hsp90-Hop interaction inhibitors

- Derry, Leigh-Anne Tracy Kim

Authors: Derry, Leigh-Anne Tracy Kim
Date: 2019
Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/63345 , vital:28395
Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
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The development of high-throughput assays to screen for potential anticancer and antimalarial compounds that target ADP-ribosylation factor 6 and its signalling machineries

- Khan, Farrah Dilshaad

Authors: Khan, Farrah Dilshaad
Date: 2019
Subjects: ADP-ribosylation , Proteins -- Metabolism , Nucleoproteins , Malaria -- Chemotherapy , Cancer -- Chemotherapy
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/92952 , vital:30810
Description: ADP-ribosylation factors (Arfs) are small GTP-binding proteins that cycle between active GTP-bound forms and inactive GDP-bound forms. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ArfGEFs activate Arfs by mediating the exchange of GDP for GTP, while ArfGAPs terminate Arf function by stimulating the hydrolysis of the terminal phosphate group of GTP. Arf6 is a major regulator of endocytic trafficking and reorganization of the actin cytoskeleton in eukaryotic organisms. Owing to its participation in wide range of fundamentally distinct cellular processes, Arf6 may be a drug target for cancer and malaria amongst other diseases. As with cancer cells, rapid growth and viability of eukaryotic pathogens likely places a heavy burden on their endocytic pathways and a critical reliance on Arf6 activity. A putative malarial homolog of Arf6 (PfArf6) localises to numerous puncta along the periphery of the parasite in the mature trophozoite life stage of the parasite (T. Swart, MSc dissertation). Owing to highly inefficient parasite transfection procedures and a relative shortage of well described and validated parasite organelle markers, the possible functions of PfArf6 were explored using HeLa cells as a surrogate model for parasites by fluorescence microscopy of cells transfected with GFP-tagged PfArf6. Partial co-localisation was observed with the mammalian markers HsArf6 and LC3, which suggested possible roles in Arf6-dependent endocytosis and autophagy, respectively. While these possible roles are currently under investigation in parasites, an overall long-term goal which was initiated in this study was to determine whether PfArf6 is a valid drug target. To chemically validate PfArf6 as a drug target, a potent inhibitor needs to be identified. This requires the development of assays that may be employed for high-throughput screening of compound libraries. To support this goal, a novel plate-based assay was developed using human Arf6. The assay relies on the selective binding of an Arf effector protein domain (GGA3) fused to glutathione-S-transferase (GST), to His-tagged Arf6 immobilised on a nickel-coated plate. The assay format was developed and could robustly distinguish HsArf6-GDP (inactive) from HsArf6-GTP (active). Furthermore, it could be employed to detect the deactivation of Arf6 by ArfGAP1-stimualted GTP hydrolysis, but not Arf6 activation by ARNO-stimulated GDP/GTP exchange (ARNO is an ArfGEF). The ArfGAP1 deactivation assay was chemically validated using a known ArfGAP inhibitor, QS11. An improved assay was developed that employs JIP4 as an Arf6-specific binding partner instead of GGA3. In addition to superior performance, the alternative assay format could potentially be exploited for cancer drug discovery, since Arf6-JIP4 interaction has been implicated in cancer cell invasion and metastasis. Both assays may be employed to explore alternative ArfGEFs and ArfGAPs that act on Arf6 and contribute to the advancement of cancer. In parallel experiments, where development of PfArf6 assays was the focus, several issues arose. Firstly, we could not prepare GDP- and GTP-bound forms of PfArf6 since EDTA-mediated nucleotide exchange appeared to irreversibly destabilise the protein. However, PfArf6 activation (i.e. the preparation of PfArf6-GTP) was possible when mediated by ARNO and assessed by tryptophan fluorescence kinetic assays, suggesting that PfArf6 may be expressed in GDP-bound form in E. coli. As with human Arf6, ARNO-mediated GDP/GTP exchange on PfArf6 was not detectable in the immobilised PfArf6-GGA interaction GST assay format. However, a more sensitive assay was developed which relies on the use of nickel-horseradish peroxidase to detect the binding of His-tagged PfArf6 to JIP4-GST immobilised on glutathione plates and could detect ARNO-mediated PfArf6 activation. Since we could not prepare PfArf6-GTP (that did not rely on the presence of the ArfGEF, ARNO), malarial ArfGAP deactivation studies were conducted using PfArf1 instead of PfArf6 in the GGA-GST interaction assay. Both PfArfGAP1and PfArfGAP2 stimulated GTP hydrolysis by PfArf1, but only the former was inhibited by the standard human ArfGAP inhibitor, QS11. The development of these simple, cost-effective assays can be used in the high-throughput screening of novel anticancer and antimalarial compounds that target Arf signalling machineries. In theory, the assay could be extended as a tool to identify novel inhibitors of the multitude of Arfs, ArfGEFs and ArfGAPs originating from any organism and hence has broad clinical significance.
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Development and optimisation of a novel Plasmodium falciparum Hsp90-Hop interaction assay

- Wambua, Lynn

Authors: Wambua, Lynn
Date: 2018
Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/62626 , vital:28216
Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
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Synthesis, characterisation and evaluation of benzoxaborole-based hybrids as antiplasmodial agents

- Gumbo, Maureen

Authors: Gumbo, Maureen
Date: 2017
Subjects: Malaria Chemotherapy , Antimalarials , Boron compounds , Drug resistance , Plasmodium falciparum , Drug development
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/59193 , vital:27456
Description: Malaria is a mosquito-borne disease, which continues to pose a threat to the entire humanity. About 40% of the world population is estimated to be at risk of infections by malaria. Despite efforts undertaken by scientific community, government entities and international organizations, malaria is still rampant. The major problem is drug resistance, where the Plasmodium spp have over the past decades developed drug resistance against available drugs. In order to counter this problem, novel antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Benzoxaborole derivatives have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported on the compounds such as 6-(2- (alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles, which showed good antimalarial activity against both W7 and 3D7 strains without significant toxicity. On the other hand, chloroquine (CQ) and cinnamic acids have a wide variety of biological activity including antimalarial activity. Herein, a hybridisation strategy was employed to synthesise new CQ-benzoxaborole and cinnamoyl-benzoxaborole hybrids. CQ-Benzoxaborole 2.12a-c and cinnamoylbenzoxaborole 2.11a-g hydrid molecules were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (1H and 13C NMR, and mass spectrometry). CQ-benzoxaborole compounds, however, showed instability, and only 2.12b was used for in vitro biological assay and showed activity comparable to CQ. Furthermore, in vitro biological assay revealed that compounds 2.11a-g poorly inhibited the growth of P. falciparum parasites. Interestingly, these compounds, however, exhibited satisfactory activity against Trypanosoma brucei with IC50 = 0.052 μM for compound 2.11g. The cell cytotoxicity assay of all final compounds confirmed that all CQ-benzoxaborole 2.12b and cinnamoyl-benzoxaborole 2.11a-g hybrids were non-toxic against HeLa cell lines. However, efforts to further expand the structure-activity relationship (SAR) of CQbenzoxaborole by increasing the length of the linker with one extra carbon (Scheme 2.10) were not possible as an important precursor 6-formylbenzoxaborole 2.29 could not be synthesized in sufficient yields. , Thesis (MSc) -- Faculty of Faculty of Science, Chemistry, 2017
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Development of a high-throughput bioassay to determine the rate of antimalarial drug action using fluorescent vitality probes

- Laming, Dustin

Authors: Laming, Dustin
Date: 2016
Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/64434 , vital:28542
Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
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Localizing selected endocytosis protein candidates in Plasmodium falciparum using GFP-tagged fusion constructs

- Basson, Travis

Authors: Basson, Travis
Date: 2016
Language: English
Type: Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/2680 , vital:20316
Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
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An investigation of the role of mitochondrial STAT3 and modulation of Reactive Oxygen Species in adipocyte differentiation

- Kramer, Adam Hildyard

Authors: Kramer, Adam Hildyard
Date: 2014
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/54632 , vital:26595
Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
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The involvement of TRAP1 in the mitochondrial localization of STAT3 in mammalian cells

- Kadye, Rose

Authors: Kadye, Rose
Date: 2014
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/55760 , vital:26731
Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
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Development of a novel, quantitative assay for determining the rate of activity of antimalarial drugs

- Khan, Tasmiyah

Authors: Khan, Tasmiyah
Date: 2013
Subjects: Assay , ATP , Antimalarials -- Therapeutic use Malaria Malaria -- Drug therapy Adenosine triphosphate Luciferases Plasmodium falciparum
Language: English
Type: Thesis , Masters , MSc
Identifier: vital:3884 , http://hdl.handle.net/10962/d1001618
Description: Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery
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